Devices and methods for dual excitation raman spectroscopy

ABSTRACT

Spectroscopic analysis systems and methods for analyzing samples are disclosed. An analysis system may contain an electromagnetic radiation source to provide radiation, a spectroscopic analysis chamber to perform a coherent Raman spectroscopy (e.g., stimulated Raman or coherent anti-Stokes Raman spectroscopy), and a radiation detector to detect radiation based on the spectroscopy. The chamber may have a resonant cavity to contain a sample for analysis, at least one window to the cavity to transmit the first radiation into the cavity and to transmit a second radiation out, a plurality of reflectors affixed to a housing of the cavity to reflect radiation of a predetermined frequency, the plurality of reflectors separated by a distance that is sufficient to resonate the radiation. The spectroscopic analysis system may be coupled with a nucleic acid sequencing system to receive a single nucleic acid derivative in solution and identify the derivative to sequence the nucleic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/675,884 filed Sep. 29, 2003, now pending; which is acontinuation-in-part application of U.S. application Ser. No. 10/262,349filed Sep. 30, 2002, now pending. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the invention relate to analyzing a sample with Ramanspectroscopy. In particular, embodiments relate to analyzing a samplecontaining a single nucleic acid derivative of interest contained withina spectroscopic analysis chamber with stimulated or coherent anti-StokesRaman spectroscopy.

2. Background Information

Deoxyribonucleic acid (DNA) sequencing has many commercially importantapplications in the fields of medical diagnosis, drug discovery, andtreatment of disease. Existing methods for DNA sequencing, based ondetection of fluorescence labeled DNA molecules that have been separatedby size, are limited by the length of the DNA that can be sequenced.Typically, only 500 to 1,000 bases of DNA sequence can be determined atone time. This is much shorter than the length of the functional unit ofDNA, referred to as a gene, which can be tens or even hundreds ofthousands of bases in length. Using current methods, determination of acomplete gene sequence requires that many copies of the gene beproduced, cut into overlapping fragments and sequenced, after which theoverlapping DNA sequences may be assembled into the complete gene. Thisprocess is laborious, expensive, inefficient and time-consuming. It alsotypically requires the use of fluorescent or radioactive labels, whichcan potentially pose safety and waste disposal problems.

More recently, methods for DNA sequencing have been developed involvinghybridization to short oligonucleotides of defined sequence, attached tospecific locations on DNA chips. Such methods may be used to infer shortDNA sequences or to detect the presence of a specific DNA molecule in asample, but are not suited for identifying long DNA sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may best be understood by referring to the followingdescription and accompanying drawings that are used to illustrateembodiments of the invention. In the drawings:

FIG. 1 shows a method for identifying a sample based on a resonanceenhanced spectroscopic analysis, according to embodiments of theinvention.

FIG. 2 shows a cross-sectional view of a spectroscopic analysis systemcontaining a chamber in a fluid channel, according to embodiments of theinvention.

FIG. 3 shows Rayleigh, Stokes, and anti-Stokes radiation.

FIG. 4 shows Stokes Raman spectra for dilute aqueous solutions of fourDNA nucleotides, according to embodiments of the invention.

FIG. 5 shows a cross-sectional view of a spectroscopic analysis systemcontaining a spheroidal chamber coupled with a fluid channel, accordingto embodiments of the invention.

FIG. 6 shows another cross-sectional view of a spheroidal chambercoupled with a fluid channel, according to embodiments of the invention.

FIG. 7A shows a view of the top of the chamber shown in FIG. 6 along thesection lines 7A-7A, according to embodiments of the invention.

FIG. 7B shows a view of the bottom of the chamber shown in FIG. 6 alongthe section lines 7B-7B, according to embodiments of the invention.

FIG. 8 shows a top-plan view of a spectroscopic analysis chamberconfigured to receive excitation radiation in the form of alignedfrequency-matched seed radiation having the same frequency as thescattered radiation, and transverse frequency-unmatched radiation havinga different frequency than the scattered radiation, according toembodiments of the invention.

FIG. 9 shows an energy level diagram for a coherent anti-Stokes RamanSpectroscopy process used to analyze a sample, according to embodimentsof the invention.

FIG. 10 shows a cross-beam configuration for implementing coherentanti-Stokes Raman spectroscopy, according to embodiments of theinvention.

FIG. 11 shows momentum conservation for a cross-beam configurationsimilar to that shown in FIG. 10, according to embodiments of theinvention.

FIG. 12 shows a forward-scattered configuration for implementingcoherent anti-Stokes Raman spectroscopy, according to embodiments of theinvention.

FIG. 13 shows a back-scattered configuration for implementing coherentanti-Stokes Raman spectroscopy, according to embodiments of theinvention.

FIG. 14 shows a nucleic acid sequencing system in which embodiments ofthe invention may be implemented.

FIG. 15 shows an exemplary apparatus and method for nucleic acidsequencing by surface enhanced Raman spectroscopy (SERS), surfaceenhanced resonance Raman spectroscopy (SERRS), resonance enhanced Ramanspectroscopy (e.g., stimulated Raman spectroscopy), and/or coherentanti-Stokes Raman spectroscopy (CARS) detection.

FIG. 16 shows the Raman spectra of all four deoxynucleosidemonophosphates (dNTPs) at 100 mM concentration, using a 100 milliseconddata collection time.

FIG. 17 shows SERS detection of 1 nM guanine, obtained from dGMP by acidtreatment according to Nucleic Acid Chemistry, Part 1, L. B. Townsendand R. S. Tipson (Eds.), Wiley-Interscience, New York, 1978.

FIG. 18 shows SERS detection of 100 nM cytosine.

FIG. 19 shows SERS detection of 100 nM thymine.

FIG. 20 shows SERS detection of 100 pM adenine, obtained from dAMP byacid treatment.

FIG. 21 shows a comparative SERS spectrum of a 500 nM solution ofdeoxyadenosine triphosphate covalently labeled with fluorescein (uppertrace) and unlabeled dATP (lower trace).

FIG. 22 shows a plot comparing the energies of Raman scattered lightgenerated from single molecules respectively contained inside an opticalresonance chamber (curves A and B) or positioned in free space withoutresonance enhancement (curve C), according to one embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are spectroscopic analysis systems and methods foranalyzing samples with Raman spectroscopy. In the following description,numerous specific details are set forth. However, it is understood thatembodiments of the invention may be practiced without these specificdetails. For example, a practitioner may use a spectroscopic analysischamber other than the specific chambers disclosed herein. As anotherexample, a practitioner may use another spectroscopy technique than thespecific stimulated and coherent anti-Stokes Raman spectroscopiesdisclosed herein. In other instances, well-known structures andtechniques have not been shown in detail in order to avoid obscuring theunderstanding of this description. It is recognized that there is agenerally high level of skill in the arts of spectroscopy and infabricating chambers as disclosed herein.

Embodiments of the invention may be used in conjunction with DNAsequencing. One exemplary method for DNA sequencing may involve theordered deconstruction of a DNA molecule into smaller nucleotidecomponents, for example through the use of enzymes, and then analyzingand identifying the nucleotides to determine the ordered sequence of thenucleotides in the original DNA molecule. Spontaneous Raman spectroscopyhas been attempted for identification of nucleotides. During spontaneousRaman spectroscopy, the sample containing the nucleotide is exposed toradiation, and a small portion of the radiation that interacts with thenucleotide is scattered due to the spontaneous Raman scattering effectaccording to vibrational characteristics of the nucleotide.

Unfortunately, in spontaneous Raman spectroscopy, the optical signalsscattered from nucleotides in dilute solution are generally very weak,and the resulting generally low probability of detecting the opticalsignals makes the approach insensitive, inaccurate, and unreliable.There have been efforts to improve the accuracy of this approach bymaking multiple measurements and performing data averaging, althoughthis takes more time, and may not be desirable in a rapid DNA sequencingenvironment. There have also been efforts to improve the strength of theoptical signal by attaching chemical moieties, such as fluorescentchromophores, to the nucleotides in order to enhance their luminescentor fluorescent properties. Reliably and consistently attaching thesemoieties remains problematic. The moieties or their buffer solutions maybe unstable. In some instances, the moieties fail to attach altogether.In other instances the moieties that do attach have differentcharacteristics and cause their nucleotides to have a differentspectroscopic response. Such problems make it difficult to accuratelyand reliably identify the nucleotides. In addition, the processes toattach the chemical moieties add additional time and complexity to theanalysis. These problems may be overcome by the methods and systemsdisclosed herein.

FIG. 1 shows a method for identifying a sample based on a resonanceenhanced stimulated Raman spectroscopic analysis, according toembodiments of the invention. The method allows identifying a samplebased on spectroscopic data that serves as a fingerprint or signaturefor the sample. In brief, the method includes adding a sample, such as asingle molecule of interest in solution, to a resonant spectroscopicanalysis chamber at block 110, analyzing the sample with a resonanceenhanced stimulated Raman spectroscopy at blocks 120-160, andidentifying the sample based on the analysis at block 170. The analysisof the single molecule is an aspect of some applications, and not alimitation. Other applications may involve analyzing a plurality ifmolecules. In some embodiments of the invention, the resonance enhancedstimulated Raman spectroscopy may include irradiating a sample containedin a resonance chamber at block 120, scattering radiation from thesample at block 130, resonating the scattered radiation in the chamberat block 140, irradiating or transmitting the scattered radiation fromthe chamber at block 150, and detecting the irradiated scatteredradiation at block 160. In one aspect, the method may be used incoordination with nucleic acid sequencing and may include identifying asingle nucleic acid derivative in a sample received from a nucleic acidsequencing system in an effort to sequence a DNA or RNA molecule.

FIG. 2 shows a cross-sectional view of a spectroscopic analysis system200 suitable to perform a resonance enhanced stimulated Ramanspectroscopic analysis of a sample in order to identify the sample,according to embodiments of the invention. The system includes anelectromagnetic radiation source 210, a fluid channel 270, aspectroscopic analysis chamber 250 (shown by dashed lines), and anelectromagnetic radiation detector 290. A sample 230 is contained withinthe chamber. During resonance enhanced spectroscopic analysis of thesample, the electromagnetic radiation source provides input orexcitation radiation 215 to irradiate the sample within the chamber inorder to generate an output of Raman inelastically scattered radiation285 that contains information that may be used to characterize andidentify the sample. In the case of stimulated Raman spectroscopy, theinput radiation may be visible, ultraviolet, or near infrared spectrumradiation from an electromagnetic radiation source such as a laser andthe output radiation may be corresponding stimulated Raman inelasticallyscattered radiation. The output radiation may be detected by thedetector and used to identify the sample or one or more componentsthereof.

Initially, with reference to block 110 of the method 100, the sample230, which may include a liquid, gas, or mixed fluid, is added to thechamber 250. In the system illustrated in FIG. 2, the sample is added tothe chamber by flowing through the fluid channel. The channel is a voidor hollowed-out space within a solid material and may be, for example,any tube, pipe, duct, or conduit to convey a fluid. The cross section ofthe channel may have a circular, oval, square, rectangular, or othershape. The fluid channel has a sample inlet 275 that is coupled with asample source to receive a sample for analysis and a sample outlet 280that is coupled with a sample destination to discharge an analyzedsample. The sample may be received at the inlet, flowed through thechannel, flowed into the chamber, analyzed, subsequently flowed out ofthe chamber, and flowed through the outlet to the proper destination.Flow may be achieved by providing an appropriate driving force, such asa pressure differential between the inlet and the outlet or a pumpwithin the channel. As one example, an aqueous solution of dilutednucleic acid derivative may be received from a pressurized nucleic acidsequencing system by pressure injection and added to the chamber byflowing through the channel in order to identify the particular nucleicacid derivative. Alternatively, the sample may be added with a fluidpump, such as a syringe, through an opening to the chamber, rather thanby flowing through a fluid channel, and subsequently removed with thesyringe, in still other implementations, if the sample is electricallycharged it may be added to the chamber under the force provided by anelectric field, or if the sample is magnetic (e.g., has a magneticmoiety attached) it may be added to the chamber by the force provided bya magnetic field.

Although not required, the channel may be micro-sized. The termmicro-sized channel, microfluidic channel, and the like will be used torefer to a channel having a cross-sectional length, for example adiameter in the case of a tubular pipe, which is less than approximatelyone millimeter (mm, one-thousandth of a meter). Often, the micro-sizedchannel may have a cross-sectional length that is in the range ofapproximately 10-500 micrometers (urn, one millionth of a meter). Tohelp put these lengths in proper perspective, the cross-sectionaldiameter of a human hair is approximately 100 micrometers. Theseminiaturized channels are often useful for handling small sized samplesand allow many channels to be constructed in a small substrate, althoughthis is not a requirement.

Often, the channel and chamber may be disposed in a solid substrate.Suitable materials for solid substrates include but are not limited toceramics (e.g., alumina), semiconductors (e.g., silicon or galliumarsenide), glasses, quartz, metals (e.g., stainless steel or aluminum),polymers (e.g., polycarbonate, polymethylmethacrylate (PMMA),polymethylsiloxine, polydimethylsiloxane (PDMS), orpolytetrafluoroethylene (Teflon®)), and combinations of these materials.Of course it will be appreciated that many other materials may also beused. Of these particular materials certain glasses, quartz, andpolymers (e.g., PMMA and polycarbonate) are known to be substantiallytransparent at least in the visible spectrum. The transparency of theseand other materials in the near infrared or ultraviolet is available inthe literature or may easily be determined without undueexperimentation. These transparent materials may be used as thesubstrate. Alternatively, non-transparent materials may be used as thesubstrate and transparent materials may be used as windows to allowsufficient transmittance of the relevant radiation. Materials that aresuitable for windows include the transparent materials previouslymentioned as well as other materials such as sapphire, magnesiumfluoride, and calcium fluoride crystals. One exemplary substratecontains a channel and cavity formed in a stainless steel substrate, thechannel and the cavity surfaces lined with an inert material such asTeflon® (for improved chemical compatibility), and one or moretransparent windows of these or other transparent materials formed intothe chamber to allow transmittance of radiation into the chamber.Alternatively, the stainless steel substrate may be replaced with glass,quartz, or ceramic, which should have sufficient compatibility withsamples that omitting the Teflon coating would be appropriate. As yetanother example, it may be desirable to use a polymer to help reducefabrication costs and take advantage of molding, hot embossing, andother fabrication techniques. An exemplary substrate may includepolydimethylsiloxane containing a channel and chamber housing molded orembossed therein into which one or more windows and reflectors may beinserted and affixed.

Various micromachining methods, such as micromilling, laser ablation,and focused ion beam milling may be used to form channels in ceramic,glass quartz, semiconductor, metal, and polymeric substrates. Approachesfor fabricating microfluidic channels are described for example in U.S.Pat. Nos. 5,904,824, 6,197,503, 6,379,974, 6,409,900, and 6,425,972.Additionally, photolithographic etches that are commonly used in thesemiconductor processing arts may be used to form channels insemiconductor, quartz, glass, and certain ceramic and metal substrates.For example, reactive plasma may be used to etch a channel having adepth of a few hundred microns and smooth vertical sidewalls in asilicon substrate. Photolithographic etches based on differentchemistries may similarly be used to form channels in polymericmaterials. In addition, molding, injection molding, stamping, hotembossing, and other approaches may be used to form channels inpolymeric materials, as well as certain metals. Other techniques thatare suitable for forming channels, as well as further backgroundinformation on microfabrication is available in “The MEMS Handbook”(ISBN/ISSN: 0849300770), by Mohamed Gad-el-Hak, published on Sep. 27,2001, containing pages 1-1368, and available from the University ofNotre Dame.

A chamber according to embodiments of the invention contains a resonantcavity to contain a sample for analysis, at least one window to thecavity to transmit a first radiation having a first frequency (e.g., aninput excitation radiation from a laser) into the cavity and to transmita second electromagnetic radiation having a second frequency (e.g., anoutput beam of stimulated Raman radiation) out of the cavity, and aplurality of reflectors (e.g., multi-layer dielectric mirrors) affixedto a housing of the cavity to reflect radiation of a predeterminedfrequency (e.g., the stimulated radiation).

Referring to FIG. 2 and the particular chamber 250, the resonant cavityis a region within the fluid channel between a reflector 235 and apartial reflector 245. The chamber has an inlet opening, at the leftmostedge of the reflectors 235, 245 (as viewed), to allow addition of asample, and an outlet opening, at the rightmost edge of the reflectors235, 245 (as viewed), to allow removal of the analyzed sample. Thesample may either be flowed continuously through the chamber or stoppedwithin the chamber. Alternatively, the chamber may be located at adead-end of the channel and may utilize a common inlet-outlet opening toadd and remove a sample.

Once the sample is contained within the chamber, with reference to block120 of the method 100 (FIG. 1), the sample is irradiated, hi the systemillustrated in FIG. 2, the source 210 provides input radiation 215 tothe chamber and irradiates the sample positioned therein. Suitablesources include coherent light sources, lasers, light emitting diodes(LEDs), lamps, and fiber optic cables coupled with such a source ofradiation. Radiation sources that provide strong monochromatic orquasimonochromatic radiation will often be favored over those thatprovide weaker or broad-spectrum radiation, since such monochromaticradiation facilitates detection of the relevant offset spectroscopicsignals. The source 210 may contain filters or monochromators to reducecertain frequencies. The source may also contain lenses, mirrors, orother devices to redirect and focus the radiation on the chamber. Thesedevices are commercially available from numerous sources, includingvendors that are listed in “The Photonics Directory™: The PhotonicsBuyers' Guide To Products and Manufacturers”. This directory containsvendor listings for radiation sources, radiation detectors,spectrometers, spectroscopic analysis software, as well as numerousother accessories (e.g., lenses, wavelength selection devices, etc.).The Photonics Directory™ is available from Laurin Publishing, and ispresently available online at the website website:www.photonics.com/directory/index.asp.

A laser (light amplification by stimulated emission of radiation) may beused to provide a high intensity beam of coherent and monochromaticlight having wavelengths suitable for resonance enhanced Ramanspectroscopy. Many different types of lasers are suitable includingtunable lasers, gas lasers, solid-state lasers, semiconductor lasers,laser diodes, VCSEL (Vertical-Cavity Surface-Emitting Laser), andquantum cavity lasers. The laser may be used to provide radiation atfrequencies or wavelengths that are suitable for Raman spectroscopy,including radiation in the visible, often in the green, red, or nearinfrared, and ultraviolet regimes. Many molecules of potential interest,including many nucleic acid derivatives, have their resonancewavelengths in or near the ultraviolet spectrum. For example, adenine, aDNA base, has a near maximum resonance wavelength at approximately 267nanometers (nm, one billionth of a meter). As such, the ultravioletspectrum as an excitation wavelength may provide comparatively strongsignals. Potential drawbacks to using ultraviolet as excitationradiation is that depending on the intensity it may damage the moleculeof interest and may induce background noise from other system componentsand compounds that also have resonance wavelengths in the ultraviolet.Lasers and associated optics for the ultraviolet range also tend to bemore costly. Another option is the use of near infrared or visibleexcitation radiation. While the signals may be slightly less intensecompared to ultraviolet, there may be comparatively less backgroundnoise, since most materials have their resonance wavelengths outside ofthese spectral frequencies. Although this particular laser is notrequired, the present inventors have utilized an argon-ion laser toprovide a radiation at about 514 nanometers wavelength and have foundthat such a laser is suitable for differentiating the Raman spectra ofDNA nucleotides. This particular laser is commercially available fromCoherent Inc. of Santa Clara, Calif. and Spectra-Physics, Inc. ofMountain View, Calif. The laser may be operated at a sufficientintensity that allows detection and avoids damaging the molecule. As anexample, the laser may be operated at energy in the range ofapproximately 100 W to 100 kW, depending upon the stability of themolecule. Other suitable lasers include a 10 mW helium-neon laserproviding radiation at a wavelength of 633 nanometers, a linearlypolarized 50 mW diode laser providing radiation in the near infrared ata wavelength of 785 nanometers, and other lasers.

Referring to FIG. 2, the chamber receives input radiation from thesource through at least one window into the cavity that allows thesample positioned therein to be irradiated. The particular chamberillustrated receives transmitted radiation 225, which is transmittedthrough the fluid channel walls, through an inlet window 220 opening tothe cavity at a left edge of the reflectors 235, 245. The inlet windowmay be a portion of the cavity housing that is at least partiallytransparent to the particular excitation radiation, a position adjacentto a reflector, a space or gap between reflectors, an incomplete orfinite reflector through which a portion of the radiation may pass, alens, an electro-optic device, or a waveguide (e.g., a fiber opticmaterial or wispering gallery mode) that confines directs and guides theradiation into or out of the cavity. Suitable electro-optic devicesinclude laser modulators and Pockel cells. A Pockel cell is a solidstate electro-optic device that is often used as a Q-switch. Anelectrical field or other signal is applied to the device in order tomodify or switch the birefringence of the cell. This allows modifyingthe transmittance of radiation through the cell and switching it on oroff Accordingly, the electro-optical device may act as a window that maybe opened or closed by applying appropriate electrical current in orderto allow transmittance of light through the device into and/or out ofthe chamber. Electro-optic devices and Pockel cells are availablecommercially from Conoptics, Inc. of Danbury Conn., among other vendors.It is also contemplated that an actual moving shutter may be opened andclosed to allow light into and/or out of the chamber. Hinged memberscommonly used in MEMS (micro-electromechanical) devices may be used forthe shutter.

As previously discussed, the transmitted input radiation 225 isintroduced into the cavity and some of the input radiation, or some ofthe reflected input radiation, or both, irradiates the sample. Withreference to block 130 of the method 100 of FIG. 1, some of theradiation is scattered by the sample. As used herein, the terms“scattered radiation” and the like will be used to refer to a photon oflight or other radiation that has collided with and been absorbed bysample matter, such as a molecule, and has been released or emitted.Most of the scattered photons will be released elastically in which thescattered radiation has no change of energy or frequency. This radiationis known as Rayleigh scattered radiation. Some of the scattered photons,often a small fraction, will be released inelastically in which there isan exchange of energy and a change of frequency. These are well-knownphenomenon. Stokes scattered radiation refers to scattering where themolecule loses energy and the frequency is reduced whereas anti-Stokesscattered radiation refers to scattering where the molecule gains energyand the frequency is increased. This scattering of light by matter isknown as Raman spectroscopy. FIG. 3 conceptually illustrates Rayleigh,Stokes, and anti-Stokes radiation. Stokes and anti-Stokes radiationtypically have lower intensity than Rayleigh radiation, and respectivelyexist at lower and higher frequencies than Rayleigh radiation. As usedherein, Stokes and anti-Stokes radiation will be referred tocollectively as inelastically scattered radiation.

The change in frequency or wavelength of the inelastically scatteredradiation is based on the particular characteristics of the sample ormolecule of interest contained therein. That is, the difference betweenthe frequencies of the input radiation and the inelastically scatteredradiation is characteristic of the sample. For example, the frequency ofa photon scattered from an inelastic collision with a molecule maydepend upon the polarization, vibration, rotation, and orientationcharacteristics of a molecule. The reasons for this are explained byquantum mechanics. Briefly, the photon may excite the molecule from itsground vibrational state to a high-energy or virtual vibrational state,from which it may relax to a lower-energy vibrational state by emissionof inelastically scattered radiation, and ultimately relax back to theground state. Further background information on this process as well asfurther background information on other vibrational spectroscopy topicsis available in “The Handbook of Vibrational Spectroscopy”, by JohnChalmers and Peter R. Griffiths, published by John Wiley & Sons, Ltd. Inany event, the frequency shifts in the light that is inelasticallyscattered by a molecule of interest incorporate information comprising afingerprint or signature for the molecules polarization, vibration,rotation, and orientation characteristics.

As previously discussed, it is often difficult and improbable to detect,or reliably detect, spontaneously scattered radiation from a dilutemolecule in solution. Accordingly, with reference to block 140 of themethod 100 of FIG. 1, the present inventors have discovered systems andmethods for resonating radiation in a cavity in order to perform aresonance enhanced spectroscopic analysis of a sample. The resonatedsignal is stronger and easier to detect than the spontaneous signal.Advantageously, this may allow improved probability and reliability ofaccurately and reliably detecting and identifying dilute molecules ofinterest in solution.

The particular chamber 250 of FIG. 2 is a resonate chamber containingthe reflector and the partial reflector to confine and resonate aradiation within the cavity in order to increase or amplify itsintensity. The inelastically scattered radiation may be resonated inorder to increase its intensity and aid detection and identification ofa sample positioned within the chamber. The reflectors may beincorporated into or affixed to the housing of the channel to internallyreflect radiation within the cavity. Radiation that is inelasticallyscattered by the sample or a portion of the input radiation 225 that isreflected by the reflector 235 may be reflected by the partial reflector245 and resonated in the cavity. Portions of the radiation that are notreflected sufficiently parallel to the optical axis normal to thereflectors may leave the chamber quickly. The reflectors may havelengths that are smaller than the distance of separation between thereflectors to allow rapid removal of misaligned radiation. In this way,in addition to resonating a particular frequency or range offrequencies, the chamber effectively selects a direction of radiationthat accumulates in the chamber, thereby allowing an intense, coherentbeam of radiation.

The cavity may have a shape and size that facilitates resonance. As-usedherein, unless specified otherwise, resonance refers to theintensification of radiation in a chamber due to reflection, and is notto be confused with excited states of electrons within a molecule. Asillustrated, chamber 250 has the reflectors opposite and parallel to oneanother, centered along a common optical axis, and separated by aparticular predetermined distance that is proportional to or at leastbased on a wavelength of radiation to be resonated in the chamber, andthat provides a non-destructive relationship between the phases ofincident and reflected radiation. When the distance between thereflectors is a multiple of approximately half the wavelength of theradiation field, the partial waves may overlap substantiallyconstructively, otherwise they may overlap less constructively ordestructively. The particular reflectors may be separated by a distancethat is approximately one half the product of the mode of the resonancefrequency, times the speed of light, divided by the index of refractionof the medium filling the cavity, divided by the frequency of theparticular radiation to be resonated in the chamber. Since coherentRaman spectroscopy may utilize excitation radiation in the visible, nearinfrared, and ultraviolet regimes, and since the inelastically scatteredradiation is merely offset from the excitation radiation, the cavity maybe designed to resonate inelastically scattered radiation havingvirtually any wavelength in the visible, ultraviolet, and near infraredregimes, depending upon the particular excitation radiation desired. Theparticular choice of excitation radiation may depend upon the cost ofthe laser and detectors, the capability of the detectors to detect aparticular radiation, the resonance and stability of the molecule ofinterest, the false signals or noise caused by other absorbing moleculesor species that are not of interest, etc.

A chamber may be designed to resonate an inelastically scatteredradiation for a molecule of interest (e.g., a particular nucleotide) orset of molecules of interest (e.g., a set of nucleotides). As oneexample, consider for a moment the Stokes spectra shown in FIG. 4 (whichwill be discussed in more detail below) generated from excitation with a514 nanometer argon-ion laser. The bottommost spectra for the moleculedeoxythymidine monophosphate has a pronounced peak at a Stokes Ramanshift of approximately 1350 cm-1. This shift is offset from theexcitation wavenumber of 19455 cm ⁻¹ (i.e., 10⁷/514=19455) for the 514nanometer excitation radiation, and translates into an actual StokesRaman wavenumber of approximately 18105 cm⁻¹ (i.e., 19455-1350). Thiscorresponds to a wavelength of 552 nanometers (i.e., 10⁷/18105). ThisStokes scattered radiation may be resonated in the chamber in order toamplify the optical signal and improve detection. The particularreflectors shown in FIG. 2 may be separated by a distance that isapproximately one half the product of the mode of the resonancefrequency times the speed of light divided by the index of refraction ofthe medium filling the cavity divided by the frequency of the particularradiation to be resonated in the chamber for this 552 nanometerscattered radiation. As another example, the chamber may be designed toresonate this wavelength of radiation as well as wavelengths ofinelastically scattered radiation from other molecules of interest. Inone embodiment of the invention, the predetermined distance between thereflectors is a function of wavelengths corresponding to prominent peaksin the Raman spectra of a plurality of molecules of interest (see e.g.,the peaks in FIG. 4). The function may be an average, a weightedaverage, or other combinatorial functions.

The reflector 235 and the partial reflector 245 may be multiple layerdielectric mirrors, metal mirrors, or other reflectors that are commonlyutilized in the spectroscopy, laser, optics, or fiber optic arts. Amulti-layer dielectric mirror, of which a Distributed Bragg Reflector(DBR) is one example, is an interference based mirror structurecontaining a stack or laminate of alternating layers of materials havingdifferent indices of refraction. The thickness of the layers may beproportional to the wavelength of the radiation to be reflected suchthat the reflected waves from the layers are in phase with one anotherand superimpose. The reflector may contain alternating layers of low andhigh refractive index materials. A dielectric or insulating material isoften used for at least one of the alternating layers and the otherlayer may be a different dielectric material, a non-dielectric material,a semiconductor, or other materials. One non-limiting example of adielectric mirror includes a plurality of alternating layers of silicon(Si) and an oxide of silicon (e.g., silicon dioxide, SiO₂), for exampleSiO₂/Si/SiO₂ that each have a thickness approximately quarterwavelength(i.e., ¼ the wavelength) of the reflected radiation (e.g., inelasticallyscattered radiation). More pairs of alternating layers may be added toimprove reflectance. The partial reflector 235 may have less totallayers than the reflector 245 in order to transmit relatively moreincident radiation than the reflector 245. Another non-limiting exampleof a dielectric mirror includes a quarterwavelength thickness of galliumarsenide (GaAs), a stress matched SiO₂/Si₃N₄/SiO₂ trilayer (alternatingdielectric layers), and about 1500 Angstroms of gold. That is, thismirror contains both a dielectric interference based mirror as well as ametal mirror. Numerous other examples of multi-layer dielectric mirrorsabound in the literature. Multi-layer dielectric mirrors may achievehigh reflectance that are often in the range of approximately 99-99.9%,or higher. These mirrors may be used to rapidly achieve good gains andincreased intensities.

A multi-layer dielectric mirror may contain layers that have a thicknessthat is proportional to a wavelength of an inelastically scatteredradiation for a molecule of interest (e.g., a particular nucleotide) orset of molecules of interest (e.g., a set of nucleotides). For example,the layers may have a thickness that is approximately quarterwavelengthof the average anti-Stokes or Stokes scattered radiation for a set ofmonobasic nucleic acid derivatives for DNA. This may allow a singleresonant cavity to be used to perform resonance enhanced spectroscopicanalysis of the entire set of derivatives, for example in the context ofDNA sequencing, and allow identification of a particular derivative ofthe set based on a resonance intensified beam incorporating derivativespecific frequency shift information that is transmitted from thecavity. As another example, the layers may have a thickness that isapproximately quarterwavelength of the 552 nanometer Stokes scatteredradiation for molecule deoxythymidine monophosphate exposed to 514nanometer radiation from an argon-ion laser. Other thicknesses may applyfor other molecules and excitation radiations.

Multi-layer dielectric mirrors are available commercially from a numberof sources or may be fabricated. SuperMirrors™ are examples of suitablemulti-layer dielectric mirrors that are available from NewportCorporation of Irvine, Calif. The SuperMirrors™ may be obtained andaffixed to the housing of the chamber. Alternatively, a multi-layerdielectric mirror may be fabricated by techniques that are commonly usedin the semiconductor processing arts (see e.g., U.S. Pat. Nos. 6,320,991or 6,208,680). As another example, a dielectric mirror containingalternating layers of Si and SiO₂ may be deposited by a conventionalchemical vapor deposition or physical vapor deposition technique that iscommonly used in the semiconductor processing arts. For example, a lowpressure chemical vapor deposition (LPCVD) technique may be used toprovides good quality layers of controlled thickness. Alternatively, aphysical vapor deposition technique such as evaporation, sputtering, ormolecular beam epitaxy may be used to deposit the layers. As anothervariation, a thicker silicon layer may be deposited by a chemical vapordeposition and a silicon dioxide layer may be thermally grown from thesilicon layer to a similar thickness. Of course, the chemical andphysical deposition techniques may also be used to deposit othermaterials.

The reflector and partial reflector may alternatively be metal mirrors.Suitable metals include, among others, aluminum, gold, silver, chrome,and alloys, mixtures, or combinations thereof. Hereinafter the termmetal will include pure metals as well as alloys, mixtures, orcombinations. Suitable metal mirrors are available from NewportCorporation. Metal mirrors may also be fabricated by conventionalchemical vapor deposition and physical vapor deposition techniques. Forexample, a gold layer may be deposited on a cavity wall by molecularbeam epitaxy evaporative deposition. Metal mirrors may providereflectance in the range of approximately 90-95%, or slightly higher butusually not greater than about 99%.

The reflected radiation may be resonated within the cavity and maystimulate or induce other inelastic scattering events. The stimulatedevents may occur more rapidly or more probabilistically than spontaneousscattering events and the intensity of the inelastically scatteredradiation may build within the chamber until saturation or equilibriumis achieved wherein round trip gain due to resonance matches loses. Itis anticipated that intensities up to on the order of a hundredmilliwatts may be obtained. Such resonance enhanced stimulated Ramanspectroscopy may provide an amplified, intense, coherent,frequency-shifted beam that should allow probable, accurate, andreliable detection and identification of even a single molecule ofinterest in dilute solution. It is not required that a single moleculeof interest be analyzed and in other embodiments a sample containing anydesired number of molecules of interest may be analyzed.

By now it should be apparent that different types of samples may beanalyzed. As used herein, the term sample will refer to one or moremolecules, atoms, or ions to be analyzed either alone or with a vapor,gas, solution, solid, or sorbent (e.g., a metal particle or colloidalaggregate). The sample may contain a dilute solution containing one ormore molecules of interest. Virtually any molecule may be of interest,depending on the particular implementation, such as a biologic molecule,a molecule associated with nucleic acid sequencing, a pharmaceutical,pesticide, an herbicide, a polymer, a pollutant, or others. For examplein the case of analyzing a biological molecule of interest associatedwith nucleic acid sequencing, an aqueous or other solution containing aprotein, protein fragment, or nucleic acid derivative may be analyzed.The term nucleic acid derivative will be used to refer to a nucleicacid, nucleic acid fragment, nucleotide, nucleoside (e.g. adenosine,cytidine, guanosine, thymidine, uridine), base (e.g. adenine, cytosine,guanine, thymine, uracil), purine, pyrimidine, or a derivative of one ofthese molecules. One exemplary sample contains a solution of a singlenucleic acid derivative having a single base that is selected from thegroup consisting of adenine, cytosine, guanine, thymine, and uracil.

Proteins provide a number of critical reactions, structures, andcontrols for cells. Nucleic acids provide cells information about whichproteins to synthesize. Nucleic acids are linear polymers of nucleotidesconnected by phosphodiester bonds. Nucleic acids may be deoxyribonucleicacid (DNA) or ribonucleic acid (RNA). Nucleotides consist of threeparts, a phosphate group, a pentose (a five carbon sugar molecule), anda base. Nucleosides are bases and sugars without a phosphate group. Thebases of both nucleotides and nucleosides are adenine (A), cytosine (C),guanine (G), thymine (T), and uracil (U). The bases adenine, cytosine,and guanine are found in both DNA and RNA, thymine usually found only inDNA, and uracil is usually found only in RNA. Accordingly, thenucleosides for DNA include deoxyadenosine, deoxyguanosine,deoxycytidine, and deoxythymidine, whereas the nucleosides for RNAinclude adenosine, guanosine, cytidine, and uridine. Likewise, thenucleotides for DNA include deoxyadenosine monophosphate, deoxyguanosinemonophosphate, deoxycytidine monophosphate, deoxythymidine monophosphate(or in the salt form, deoxyadenylate, deoxyguanylate, deoxycytidylate,and thymidylate), whereas the nucleotides for RNA include adenosinemonophosphate, guanosine monophosphate, cytidine monophosphate, uridinemonophosphate (or in the salt form, adenylate, guanylate, cytidylate,and uridylate). In RNA the pentose is ribose whereas in DNA the pentoseis deoxyribose. The bases cytosine, thymine, and uracil are pyrimidinebases and contain a single heterocyclic ring containing carbon and otheratoms, in this case nitrogen. The bases adenine and guanine are purinebases and contain a pair of fused heterocyclic rings that also containnitrogen. The ability to determine a nucleic acid and/or proteinsequence may have tremendous commercial importance in the fields ofmedical diagnosis, treatment of disease, and drug discovery. Of course,these are just examples of the types of samples and molecules that maybe analyzed by the systems and methods developed by the presentinventors.

With continued reference to the method 100 of FIG. 1, and to block 150,after resonating, the scattered radiation may be irradiated ortransmitted from the chamber. Referring to FIG. 2, the chamber maycontain at least one window to allow the radiation to exit the chamberand be detected. The particular chamber 250 contains an outlet window260, which in this particular instance comprises the partial reflector245. The partial reflector is an incomplete or finite reflector that hasa sufficient reflectivity to achieve resonance and a sufficienttransmittance to allow a detectable level of incident radiation to betransmitted to the detector 290. The reflector 235 may have a highertotal reflectance than the partial reflector 245 in order to providegood amplification of intensity in the cavity, although this is notrequired. In one instance, the reflector 235 may have a highreflectivity for both the input excitation radiation and theinelastically scattered radiation, for example greater thanapproximately 99% (e.g., approximately 99.9%) for both, whereas thepartial reflector 245 may have a sufficient transmittance for theinelastically scattered radiation, for example a transmittance that isnot less than approximately 1% (or a reflectance that is not greaterthan approximately 99%). Of course alternate types of outlet windows mayalso be used, such as a transparent material incorporated into thecavity housing, a Pockel cell, a space, gap, slit, pinhole, or otheropening in the reflector 235, or a shutter. Additionally, as anotheralternative, a single inlet-outlet window may be utilized to transmitradiation into and out of the chamber.

At least a portion of the resonated radiation 240 that is incident onthe partial reflector 245 leaves the cavity and chamber as outputradiation 285. The output radiation contains inelastically scatteredradiation that incorporates frequency-shift information about the samplein the chamber. The intensity of the output radiation may depend uponthe reflectivity of the partial reflector, the resonance characteristicsof the chamber, and upon losses due to absorption, scattering anddiffraction. Often, the chamber will be designed to achieve highintensities of the output radiation to allow accurate detection andidentification of samples.

With reference again to the method 100 of FIG. 1, at least some of theinelastically scattered radiation in the output radiation 285 isdetected at block 160. The electromagnetic radiation detector 290 isshown in simplified format and is to be interpreted broadly. Often, theoutput radiation may be passed through a lens and a wavelength selectiondevice. The lens may collect, direct, and focus the radiation emittedfrom the chamber. The light from the lens or from the chamber may bepassed through a wavelength selection device that emphasizes, selects,separates, or isolates a particular frequency or range of frequencies.The wavelength selector may be used to distinguish radiation of interest(e.g., inelastically scattered radiation) from other radiation (e.g.,elastically scattered radiation and excitation radiation). Suitablewavelength selection devices include among others prisms,monochromators, filters (e.g., absorbance, bandpass, interference, orFourier), dichroic filters, dichroic mirrors, and demultiplexers.

After passing the radiation from the chamber through any lenses,wavelength selection devices, and the like, the resultant radiation maybe passed to a spectrometer, optical multichannel analyzer (OMA), orother radiation detection device. These radiation detection devices willoften employ an optical transducer to convert received radiation into acorresponding electrical signal that captures and represents at leastsome of the same information. Exemplary optical transducers include acharge coupled device (CCD), phototransistor, photomultiplier tube,photo diode, or an array of one or more of these devices.

The radiation detection device may comprise a Raman spectrometer.Spectrometers are well known radiation detection devices that measurethe wavelength and intensity of light. Spectrometers are commerciallyavailable from a number of sources. One suitable Raman spectrometer isSpectraPro 300i Spectometer available from Acton Research Corporation ofActon Mass. Another spectrometer that is suitable is the RAMANRXN1Analyzer available from Kaiser Optical Systems, Inc. (website:www.kosi.comi) of Ann Arbor, Mich. This spectrometer uses athermoelectrically cooled Charge Coupled Device (CCD) array to providehigh sensitivity detection of radiation. Optionally, this spectrometermay also be obtained with an Invictus NIR Laser as a radiation source, aSuperNotch® Filter, a Holographic Laser Bandpass Filter, and a HoloSpecholographic imaging spectrograph. Further background information aboutthis spectrometer including its operation is available from the vendor.Of course other spectrometers may also be used.

Often, the spectrometer may generate spectra showing frequency orwavelength versus intensity. FIG. 4 shows Stokes Raman spectra forhighly diluted aqueous samples of each of the four DNA nucleotides,according to embodiments of the invention. For clarity, the four spectrahave been offset or displaced from one another along the vertical axis.From top to bottom, the spectra are for deoxyadenosine monophosphate,deoxycytidine monophosphate, deoxyguanosine monophosphate, anddeoxythymidine monophosphate. The spectra were generated by performingspontaneous Raman spectroscopic analysis on the samples includingirradiating the samples with 514 nanometer radiation from a laser anddetecting the output inelastically scattered radiation with a Ramanspectrometer. The spontaneous spectra should be substantially similar toresonance enhanced Raman spectra except that the signals would becorrespondingly weaker in intensity. The figure shows intensity inarbitrary units versus Raman wavelength shift (Stokes offset from theexcitation radiation) in reciprocal centimeters. As shown, each of thespectra have distinct spectral characteristics, prominent peaks, orfingerprint bands, that identify the corresponding nucleotide. Exemplaryprominent peaks include 1630 cm⁻¹ for the top spectra, 1440 cm⁻¹ for thenext spectra down, 800 cm⁻¹ for the second spectra from the bottom, and1350 cm⁻¹ for the bottom spectra.

Finally, with reference to the method 100 of FIG. 1, the sample may beidentified at block 170. The sample may be identified by comparing thedetermined spectrum with a spectral library containing manypredetermined spectrum for known samples and identifying the sample asone of the known samples if the determined spectrum sufficientlyapproximates the corresponding predetermined spectrum in the library.The electrical signals generated by the radiation detector as a resultof the output radiation may be provided to a computer system that isappropriately programmed with spectroscopic analysis instructions andthe library (e.g., a database) that allows the Raman fingerprint orsignature represented in the electrical signals to be correlated to aspecific fingerprint or signature in the library. For example, in thecase of a spectrometer, a spectrum (or certain bands thereof) for asample may be compared or contrasted to spectroscopic data for otheridentified samples to determine whether the fingerprints aresufficiently identical (i.e., the sample has the same identity as theidentified sample in the database). Various methods for identificationof nucleotides by Raman spectroscopy are known in the art (see e.g.,U.S. Pat. Nos. 5,306,403, 6,002,471, or 6,174,677). The identity of thesample may be stored in memory, further analyzed, or used for otherpurposes.

With reference again to the method 100 of FIG. 1, another way ofimplementing this method may include adding a sample to a chamber atblock 110, irradiating the sample with a short and accurately-knownpulse of a radiation having substantially the same frequency orwavelength as an inelastically scattered wavelength relevant to thesample at block 120, then scattering, resonating, and irradiating theradiation from the cavity at blocks 130-150, respectively, and thendetecting the radiation as well as some indication of the decay time ofthe inelastically scattered light in the chamber at block 160. The decaytime may be used as a fingerprint or signature for the identity of thesample. Of course other methods are also contemplated.

FIG. 5 shows a cross-sectional view of a spectroscopic analysis system500 suitable to perform a resonance enhanced spectroscopic analysis of asample in order to identify the sample, according to embodiments of theinvention. The system includes a radiation source 510, a substrate 505,a fluid channel 570, a spectroscopic analysis chamber 550, and aradiation detector 590. A sample 530 is contained within the chamber.The channel and the chamber are formed within the substrate and arecoupled. The particular chamber contains a spheroidal resonant cavity,an inlet window 520, a first, second, third and fourth curved reflectors535A-D, and an outlet window 560. The sample may be added to the channelat an inlet 575 thereof, then flowed into a cavity of the chamber andspectroscopically analyzed, and then flowed out of the chamber andthrough the sample outlet 580.

The spheroidal resonant cavity is formed of concave surfaces that mayassist with reflecting radiation toward the interior of the cavity. Theterm spheroidal will be used to refer to a shape that resembles orapproximates a sphere but that is not necessarily a perfect sphere. Ofcourse, the spheroidal shape is not required and in other cavities onlya portion of the chamber housing may be concave (e.g., spherically orparabolically concave), or the cavity may be a cylindrical void (e.g.,having a diameter greater than that of the fluid channel), a polyhedronvoid, a hexahedron void, a cubic void, or a void having any otherregular or irregular shape.

The cavity serves as a wide spot in the fluid channel and may have avolume that is convenient for the particular implementation. Typically,the cavity will have a volume that is not greater than about amilliliter (mL, one-thousandth of a liter). Alternatively, if a smallercavity is favored, the cavity may be a micro-sized cavity or microcavityhaving a volume that is not greater than approximately a microliter (μL,one-millionth of a liter). For example, the volume may be approximately500 nanoliters (nL, one billionth of a liter), approximately 100 nL,approximately 1 nL, or less.

An input excitation radiation 515 from the source 510 is directedthrough the substrate, through the inlet window 520, and into thecavity. The particular inlet window 520 comprises a region of the cavityhousing between the second and third reflectors 535B-C that is at leastpartially transparent to the inlet radiation 515. The inlet window maybe a space between the second and third reflectors, for example aportion of sufficiently transparent substrate, or a partial reflectorbetween the second and third reflectors or a transparent material. Atleast some of the input radiation irradiates the sample and isinelastically scattered. The reflectors 535A-D confine the inelasticallyscattered radiation, and in this particular embodiment much of the inputradiation, inside the chamber by internal reflection. Some of thereflected radiation may stimulate further inelastically scatteredradiation by re-irradiating the sample and again being inelasticallyscattered. In another embodiment of the invention, such as thatdiscussed in FIG. 8, an outlet window may be used to remove the inputexcitation radiation from the cavity.

Output radiation 585 leaves the cavity through the outlet window 560 andcontains at least some resonance enhanced stimulated inelasticallyscattered radiation. The outlet window is positioned away from the pathof the input radiation, and primary reflections thereof, to reducetransmission of the inlet radiation through the outlet window. Ofcourse, this is not required, and a wavelength selection device may beused to remove any such radiation that is transmitted.

FIG. 6 shows another cross-sectional view of a spheroidal chamber 650,according to embodiments of the invention. The chamber shows thefeatures of the chamber 550 as well as alternate locations 676, 677,678, and 679 for either the inlet window 620 or outlet window 660. Byway of example, the outlet window may be re-positioned at one of thelocations 676-679, or the inlet window may be re-positioned at one ofthe locations 676-679. A sample 630 is positioned within the chamber.FIG. 6 also shows section lines 7A-7A and 7B-7B that respectively showthe views of FIGS. 7A and 7B.

FIGS. 7A-7B show fabrication of the chamber 650 of FIG. 6, according toembodiments of the invention. FIG. 7A shows a top-plan view of the topof the chamber 650 along the section lines 7A-7A shown in FIG. 6. Thehemi-spheroid void 750A of the top of the chamber 650 and thehemi-cylindrical voids 770A (half of a cylindrical channel void) may beformed within a substrate 705A. In one example, these voids 750A, 770Amay be formed in a quartz or glass substrate by photolithography andetching. An anisotropic etch with agitation may be used to form thenearly spherical concave surfaces of the chamber 750A and the concavecylindrical surfaces of the channel 770A by etching along crystalplanes. In another example, these voids may be formed in a polymericmaterial, such as PDMS by molding or embossing.

The interior surface of the hemi-spheroid void 750A contains reflectors735B-D (which correspond to the reflectors 635B-D in the view of FIG.6). The reflector may be formed by subsequent alternating deposition oflayers of appropriate thickness of materials having different index ofrefraction (e.g., Si and SiO₂). The depositions may be over the wholehemisphere in which case an inlet window 720 and an exit window 760 maybe formed by removing a portion of these deposited layers to form apartial reflector, or by removing all of these layers to form atransparent quartz window. These layers may be removed by laserablation, by etching, or by other conventional techniques for removinglayers. Alternatively, a mask patterned with the sizes and positions ofthe windows 720 and 760 may be used to selectively deposit themulti-layer dielectric mirror layers over the non-window portions. Inthe case of the PDMS polymer, a sapphire, quartz, or other transparentwindow may be molded into the polymer or the polymer drilled and thewindow fixedly inserted.

FIG. 7B shows a top-plan view of the bottom of the chamber 650 along thesection lines 7B-7B shown in FIG. 6. The hemi-spheroid void 750B of thebottom of the chamber 650, the hemi-cylindrical voids 770B, and thereflector 735B may be formed within a substrate 705B, such as a siliconsubstrate, as described for FIG. 7A. After forming these structures, thesubstrate 705A may be bonded to the substrate 705B by a wafer bondingapproach, for example by a high temperature fusion of oxidized surfacesthereof, or with an adhesive.

FIG. 8 shows a top-plan view of a spectroscopic analysis chamber 850configured to receive input excitation radiation in the form of alignedseed radiation 815B and transverse radiation 815A, according toembodiments of the invention. The input transverse excitation radiationmay have any suitable frequency. The direction of the transverseradiation is crosswise, perpendicular, to the direction of the outputradiation 885, and to the direction of resonance. This allows a portionof the transverse radiation 886 that does not irradiate a molecule 831,to leave the chamber. This may aid in detecting the output inelasticallyscattered radiation 885. The seed radiation is added to the chamberthrough a partial reflector 845. The seed radiation may have the samefrequency as radiation inelastically scattered from a particular sampleand may stimulate scattering if that sample is contained within thechamber. The direction of the seed radiation is opposite, but alignedwith, the direction of output inelastically scattered radiation 885.This allows the seed radiation transmitted through the partial reflectorto be reflected and resonated. The radiation detector may be configuredto determine the gain or increase in intensity of the output radiationover the known intensity of the input seed radiation, which gain orincrease may be attributed to stimulated scattering.

Accordingly, with continued reference to the method 100 of FIG. 1,irradiating a sample at block 110 may include irradiating a sample withan input transverse radiation and an input seed radiation havingdifferent frequencies. Likewise, detecting radiation at block 160 mayinclude detecting a gain over the intensity of the seed radiation. Theseed radiation frequency may be varied over a set of predeterminedfrequencies corresponding to different molecules to determine whetherthe sample within the chamber contains a molecule corresponding to oneof these frequencies. For example, the seed radiation may be provided atan inelastically scattering frequency for adenine. If the gain over theseed radiation is sufficiently zero then there was no simulatedscattering and it may be concluded that the sample did not containadenine. Then the frequency of the seed radiation may be adjusted tothat corresponding to other nucleotide bases until the gain issufficiently nonzero indicating that the sample may contain thatparticular base. If none of the frequencies cause the gain to becomesufficiently nonzero, the sample may be removed from the chamber andanother sample added thereto.

The concepts of seed and transverse input radiations discussed in FIG. 8may also be applied to a spectroscopic analysis system similar to theones shown in FIGS. 5-6. For example, input transverse excitationradiation may be transmitted in through window 620, input seed radiationmay be transmitted in through window 660, and scattered radiation may betransmitted out through window 660. Portions of the input transverseradiation that do not irradiate the sample may be removed through awindow at location 679.

Accordingly, in embodiments of the invention employing a stimulatedRaman analysis, a single molecule of interest may be positioned within aresonant chamber that is capable of optically amplifying the intensityof inelastically scattered stimulated Raman radiation by causingresonance enhanced stimulated scattering events that aid in increasingor amplifying the intensity of the signal to an extent that allowsdetection and accurate identification of the single molecule ofinterest. As an example, a single nucleic acid derivative of interestmay be positioned in a resonant chamber having two opposing reflectors.The first reflector has a high reflectivity for both an excitationradiation and an inelastically scattered Raman radiation, while thesecond reflector has a high reflectivity for the excitation radiationand has a sufficient transmittance for the inelastically scattered Ramanradiation. In one aspect, a high reflectivity may be greater thanapproximately 99% (e.g., 99.5%-99.9%) while a sufficient transmittancemay be a reflectivity not greater than 99%. The two opposing reflectorsare separated by a distance that is sufficient to resonate theinelastically scattered Raman radiation. The distance may be determinedbased on inelastically scattered Raman radiation for the single nucleicacid derivative, or for a plurality of nucleic acid derivatives so thatthe chamber may be used for identifying different derivatives.

Accordingly, a sample containing a single molecule of interest may beanalyzed with stimulated Raman spectroscopy. In addition to stimulatedRaman spectroscopy, another suitable form of coherent Raman spectroscopyis Coherent Anti-Stokes Raman Spectroscopy (CARS), which employs acoherent anti-Stokes Raman scattering phenomenon. Many other forms ofRaman, such as spontaneous Raman and surface enhanced Raman spectroscopy(SERS), involve spontaneous emission of random and non-coherentinelastically scattered radiation. In contrast, CARS inherently producesa highly directional and coherent output inelastically scatteredradiation signal. Rather than due to resonance, as in stimulated Raman,the coherency is provided by coherent build up of amplitudes for phasematched radiations. As used herein, CARS will encompass variousimplementations of CARS, such as time-resolved CARS, scanned CARS, OPOCARS, MAD CARS, PARS, and others. The CARS radiation signal issufficiently coherent and intense to allow detection of even a singlemolecule of interest. The spectral shape and intensity of the CARSoutput signal contains the spectral characteristics of the sample andmay be used to identify the sample. Accordingly, the present inventorscontemplate employing CARS to analyze and identify a single nucleic acidderivative in conjunction with nucleic acid sequencing.

CARS is a four-wave mixing spectroscopy. During CARS a sample in achamber is irradiated and excited with a first radiation having a firstwavelength (w₁) and often a second radiation having a second wavelength(W₂) that overlap and are phase matched (i.e., they spatially andtemporally overlap) in order to induce the sample to irradiate coherentanti-Stokes scattered radiation having a third wavelength (W₃) that isrelated to the first and second frequencies according to therelationship, W₃=2w₁−W₂.

FIG. 9 shows an energy diagram for a CARS process used to analyze amolecule of a sample, according to embodiments of the invention. Energyof the molecule is plotted on the y-axis and the progression of the CARSprocess is plotted on the x-axis. For convenience, the discussion willrefer to a photon, although often in practice a radiation containing atleast one but often many photons will be used to analyze the sample.Initially, the molecule is excited coherently with a first photon atwavelength (w₁) 910 in order to induce an emission of a second photon atwavelength (w2) 920. The first photon is often provided as a laser beampulse directed at the chamber to irradiate the molecule. As shown, thefirst photon excites the molecule from a ground vibrational state 950 toa lower virtual state 970. The emitted second photon brings the moleculefrom the virtual state 870 down to a lower excited vibrational state960. The excited vibrational state differs from the ground vibrationalstate by vibratory transition of Raman shift (W₄). The molecule may emitthe second photon by either spontaneous or stimulated emission howevercoherent stimulated emission is used in CARS. Often, the molecule willbe exposed by another photon also of wavelength (W₂) in order to inducestimulated emission of the second photon. Stimulated emission isrelatively more probable than spontaneous emission and may lead tohigher CARS signal intensities that facilitate detection and moleculeidentification. The stimulated emission is often induced by irradiatingthe chamber with a second radiation having the second wavelength (W₂),concurrently with the irradiation with the first radiation (the firstphoton).

The molecule in the lower excited vibrational state 960 is subsequentlyirradiated with a third photon at wavelength (w₁) 930. Irradiation witha laser beam pulse may also provide this photon. The third photonexcites the molecule from the excited vibrational state 960 to a highervirtual state 980. The molecular vibrations oscillate in phase andinterfere constructively. The molecule emits a fourth photon atwavelength (W₃) to drop from the higher virtual state to the groundvibrational state. The wavelength of the fourth photon is twice thewavelength of the first and third photons minus the wavelength of theemitted second photon (i.e., w₃=2w1−w₂). In essence, CARSdisproportionates or divides two (w₁) photons into a (w₂) photon and a(W₃) photon. A phase matching condition determines the direction of thecoherent CARS signal. As will be discussed further below, the presentinventors contemplate employing various geometric configurations ofbeams to take advantage of this property to help spatially isolate thecoherent CARS signal from input excitation beams, in order to facilitatedetection and identification of molecules. The intensity of the emittedCARS signal is directly related to the intensity of the excitationradiations.

Different ways of exposing the sample to these photons are known in thearts. In one approach, the sample may be concurrently irradiated with afirst laser beam pulse of the wavelength w₁, a second laser beam pulseof the wavelength W₂, and a third laser beam pulse of the wavelength(w₁). In practice, the first laser beam pulse can also serve as a thirdlaser beam pulse as they have the same wavelength, (w₁). Electronics toprovide concurrent laser beam pulses from multiple lasers arecommercially available. Such synchronization electronics includeSynchroLock from Coherent (Santa Clara, Calif.) or Lok-to-Clok fromSpectra-Physics (Mountain View, Calif.). Alternatively, in anotherapproach the sample may be continually irradiated with a laser beam ofthe wavelength (w₁) and intermittently irradiated with a pulse or scanof a laser beam of the wavelength (w₂). When the difference (w₁−w₂)substantially coincides or matches the wavelength of a molecularvibration of the sample, for example the vibratory transition (w₄), theintensity of the CARS signal is often significantly enhanced. A spectrummay be generated for a range of the wavelength (w₂) or the difference(w₁−w₂). Often, (w₂) is continually changed, varied, or scanned, whileholding (w₁) fixed, and simultaneously recording the corresponding CARSsignals. Alternatively, (w₁) is continually changed, varied, or scanned,while holding (w₂) fixed, and simulateneously recording thecorresponding CARS signals. In another embodiment, both (w₁) and (w₂)are continually changed, varied, or scanned, to obtain the desired(w₁−w₂), while recording the corresponding CARS signals. In one approacha tunable broadband dye laser is scanned over the desired range ofwavelength. Another approach involves using a broadband Stokes lasercovering the whole range of frequencies concurrently. Alternatively,signal and idler beams from optical parametric oscillator can be used toscan (w₁−w₂).

In order for a CARS process to occur efficiently energy and momentumconservation need to occur. During CARS the output signal is built bycoherent addition of amplitudes. In the CARS four-wave mixing process,it will often be desirable to impose a phase matching condition on thewave vectors of the input excitation radiations and output coherentanti-Stokes radiation in order to achieve good intensities of the outputcoherent radiation. The amplitudes should be substantially in phase,that is the beams aligned to provide phase matching between the sum oftwo incoming waves at wavelength (w₁) and the sum of output waves atwavelength (w₂) and (w₃). When the phase matching condition issatisfied, the strength or intensity of the output anti-Stokes waveshould be near maximum. This results in the sample irradiating oremitting a coherent resonance enhanced anti-Stokes scattered radiation,often as a coherent, collimated, highly-directional beam with apredetermined direction, rather than as a weak and randomly scatteredsignal as would be expected in spontaneous Raman spectroscopy. Thepredetermined direction of the CARS signal may allow variousconfigurations that allow it to be geometrically or configurationallyseparated from the excitation radiation.

A number of prior art phase matching beam configurations or geometriesare known in the arts including a collinear CARS geometry, a BOX CARSgeometry, a Folded BOX CARS geometry, and a USED CARS geometry. Any ofthese may potentially be used. Additionally, the inventors contemplateusing configurations that help separate or isolate the CARS radiationsignal from input excitation radiation. FIGS. 11-13 show exemplaryconfigurations of excitation radiation beams that when phase matched mayhelp to spatially separate and isolate the CARS signal from theexcitation beams at the radiation detection end, according to variousembodiments of the invention. Advantageously, in these exemplaryconfigurations, the CARS signal beam is spatially separated and isolatedfrom the excitation beams at the radiation detection end, which mayfacilitate sample identification. Other configurations will be apparentto those having an ordinary level of skill in the art and the benefit ofthe present teachings.

FIG. 10 shows a cross-beam configuration, according to embodiments ofthe invention. In the cross-beam configuration, an input first (w₁) andsecond radiation (w₂) are provided to a chamber 1050 with an anglerelative to one another so that they irradiate a sample 1030 within thechamber and subsequently separate from one anther and from a coherentanti-stokes scattered radiation (w₃) that is irradiated from the sample.In this way, the configuration helps separate the input radiation fromthe output coherent scattered radiation, and wavelength selection orfiltering may be avoided. The chamber may be similar to other chambersdisclosed herein. The chamber may, but need not, be a resonant chamberand may, but need not, contain reflectors or have a size or shape tofacilitate resonance.

FIG. 11 shows momentum conservation for a cross-beam configurationsimilar to that shown in FIG. 10. The phase matching conditiondetermines the direction of the coherent anti-Stokes radiation. Becauseof the conservation of energy and momentum in a four-wave mixingprocess, a phase matching condition is imposed on the wave vectors ofthe input and anti-Stokes output waves. When the phase matchingcondition is sufficiently satisfied the output strength is near maximumand the direction and momentum of the coherent anti-Stokes photons arerelated to the direction and momentum of the excitation photons. This isshown graphically in FIG. 11 wherein two vectors for the firstexcitation wavelength (2k_(w1)) and one vector for the second excitationwavelength (k_(w2)) are combined to give a vector for the coherentanti-Stokes radiation (k_(w3)), or mathematically k_(w3)=2k_(w1)−k_(w2).The length of the vectors represents the photons momentum. Theconfiguration of the direction of the excitation radiations allows theCARS signal photon to be geometrically separated from the inputexcitation photons. This may facilitate detection of the emitted CARSphoton.

FIG. 12 shows a forward-scattered configuration, according toembodiments of the invention. In this configuration, the coherentradiation is not separated from the input radiations, and a wavelengthselection device, such as a filter, may be used to isolate the coherentradiation.

FIG. 13 shows a back-scattered configuration, according to embodimentsof the invention. In this configuration, as in the cross-beamconfiguration, the coherent radiation is separated from the inputradiations. The intensity of the coherent radiation in thisconfiguration is often comparable with that of the forward-scatteredconfiguration when the sample size is less than the excitationwavelength, although in other instances it may be desirable to use aforward-scattered configuration and appropriate wavelength selectiondevice to obtain a stronger signal.

FIG. 14 shows a nucleic acid sequencing system 1401 in which embodimentsof the invention may be implemented. A nucleic acid may be provided to asampling system 1402. The sampling system may include enzymes or othercleavers of nucleic acids to remove or derive a single nucleic acidderivative from the nucleic acid. In one instance, the system mayinclude an exonuclease enzyme that breaks down a nucleic acid chain byremoving single nucleotides one by one from the end of a chain. Thesampling system may also include equipment to prepare the sample, forexample to dilute the nucleic acid derivative in an aqueous solution andsupply any additives. The sample may be added to the spectroscopicanalysis system 1400, for example though a fluid channel 1470. Theanalysis system performs a Raman spectroscopic analysis of the sample,as described elsewhere herein, in order to identify the nucleic acidderivative. For example, the analysis system may determine whether thesample contains a single base selected from the group comprisingadenine, cytosine, guanine, thymine, and uracil. The identity of thenucleic acid derivative may be stored in a memory as part of an orderedsequence for the input nucleic acid, analyzed as part of the sequence tosolve a medical problem, for example to diagnose or treat a disease, orused for other purposes.

Having been generally described, the following examples are given asparticular embodiments of the invention, to illustrate some of theproperties and demonstrate the practical advantages thereof, and toallow one skilled in the art to utilize the invention. It is understoodthat these examples are to be construed as merely illustrative.

EXAMPLES Example 1 Nucleic Acid Sequencing Using Raman Detection andNanoparticles

Certain embodiments of the invention, exemplified in FIG. 15, involvesequencing of one or more single-stranded nucleic acid molecules 1509that may be attached to an immobilization surface in a reaction chamber1501. The reaction chamber 1501 may contain one or more exonucleasesthat sequentially remove one nucleotide 1510 at a time from theunattached end of the nucleic acid molecule 1509.

As the nucleotides 1510 are released, they may move down a microfluidicchannel 1502 and into a nanochannel 1503 or microchannel 1503, past adetection unit. The detection unit may comprise an excitation source1506, such as a laser, that emits an excitatory beam. The excitatorybeam may interact with the released nucleotides 1510 so that electronsare excited to a higher energy state. The Raman emission spectrum thatresults from the return of the electrons to a lower energy state may bedetected by a Raman spectroscopic detector 1507, such as a spectrometer,a monochromator or a charge coupled device (CCD), such as a CCD camera.

The excitation source 1506 and detector 1507 may be arranged so thatnucleotides 1510 are excited and detected as they pass through a regionof nanoparticles 1511 in a nanochannel 1503 or microchannel 1503. Thenanoparticles 1511 may be cross-linked to form “hot spots” for Ramandetection. By passing the nucleotides 1510 through the nanoparticle 1511hot spots, the sensitivity of Raman detection may be increased by manyorders of magnitude. Alternatively, the nucleotides may be passed into aresonant chamber.

Preparation of Reaction Chamber, Microfluidic Channel and MicroChannel

Borofloat glass wafers (Precision Glass & Optics, Santa Ana, Calif.) maybe pre-etched for a short period in concentrated HF (hydrofluoric acid)and cleaned before deposition of an amorphous silicon sacrificial layerin a plasma-enhanced chemical vapor deposition (PECVD) system (PEII-A,Technics West, San Jose, Calif.). Wafers may be primed withhexamethyldisilazane (HMDS), spin-coated with photoresist (Shipley 1818,Marlborough, Mass.) and soft-baked. A contact mask aligner (QuintelCorp. San Jose, Calif.) may be used to expose the photoresist layer withone or more mask designs, and the exposed photoresist may be removedusing a mixture of Microposit developer concentrate (Shipley) and water.Developed wafers may be hard-baked and the exposed amorphous siliconremoved using CF₄ (carbon tetrafluoride) plasma in a PECVD reactor.Wafers may be chemically etched with concentrated HF to produce thereaction chamber 1501, microfluidic channel 1502 and microchannel 1503.The remaining photoresist may be stripped and the amorphous silicon maybe removed.

Nanochannels 1503 may be formed by a variation of this protocol.Standard photolithography may be used to form the micron scale featuresof the integrated chip. A thin layer of resist may be coated onto thechip. An atomic force microscopy/scanning tunneling probe tip may beused to remove a 5 to 10 nm wide strip of resist from the chip surface.The chip may be briefly etched with dilute HF to produce a nanometerscale groove on the chip surface. In the present non-limiting example, achannel 1503 with a diameter of between 500 nm and 1 μm may be prepared.

Access holes may be drilled into the etched wafers with a diamond drillbit (Crystalite, Westerville, Ohio). A finished chip may be prepared bythermally bonding two complementary etched and drilled plates to eachother in a programmable vacuum furnace (Centurion VPM, J. M. Ney,Yucaipa, Calif.). Alternative exemplary methods for fabrication of achip incorporating a reaction chamber 1501, microfluidic channel 1502and nanochannel 1503 or microchannel 1503 are disclosed in U.S. Pat.Nos. 5,867,266 and 6,214,246. A nylon filter with a molecular weightcutoff of 2,500 daltons may be inserted between the reaction chamber1501 and the microfluidic channel 1502 to prevent exonuclease and/ornucleic acid 1509 from leaving the reaction chamber 1501.

Nanoparticle Preparation

Silver nanoparticles 1511 may be prepared according to Lee and Meisel(J. Phys. Chem. 86:3391-3395, 1982). Gold nanoparticles 1511 may bepurchased from Polysciences, Inc. (Warrington, Pa.), Nanoprobes, Inc.(Yaphank, N.Y.) or Ted-pella Inc. (Redding, Calif.). In a non-limitingexample, 60 nm gold nanoparticles 1511 may be used. The skilled artisanwill realize that other sized nanoparticles 1511, such as 5, 10, or 20nm, may also be used.

Gold nanoparticles 1511 may be reacted with alkane dithiols, with chainlengths ranging from 5 nm to 50 nm. The linker compounds may containthiol groups at both ends of the alkane to react with gold nanoparticles1511. An excess of nanoparticles 1511 to linker compounds may be usedand the linker compounds slowly added to the nanoparticles 1511 to avoidformation of large nanoparticle aggregates. After incubation for twohours at room temperature, nanoparticle 1511 aggregates may be separatedfrom single nanoparticles 1511 by ultracentrifugation in 1 M sucrose.Electron microscopy reveals that aggregates prepared by this methodcontain from two to six nanoparticles 1511 per aggregate. The aggregatednanoparticles 1511 may be loaded into a microchannel 1503 bymicrofluidic flow. A constriction or filter at the end of themicrochannel 1503 may be used to hold the nanoparticle aggregates 1511in place.

Nucleic Acid Preparation and Exonuclease Treatment

Human chromosomal DNA may be purified according to Sambrook et al.(1989). Following digestion with Bam H1, the genomic DNA fragments maybe inserted into the multiple cloning site of the pBluescript® IIphagemid vector (Stratagene, Inc., La Jolla, Calif.) and grown up in E.coli. After plating on ampicillin-containing agarose plates a singlecolony may be selected and grown up for sequencing. Single-stranded DNAcopies of the genomic DNA insert may be rescued by co-infection withhelper phage. After digestion in a solution of proteinase K:sodiumdodecyl sulphate (SDS), the DNA may be phenol extracted and thenprecipitated by addition of sodium acetate (pH 6.5, about 0.3 M) and 0.8volumes of 2-propanol. The DNA containing pellet may be resuspended inTris-EDTA buffer and stored at −20° C. until use.

M13 forward primers complementary to the known pBluescript® sequence,located next to the genomic DNA insert, may be purchased from MidlandCertified Reagent Company (Midland, Tex.). The primers may be covalentlymodified to contain a biotin moiety attached to the 5′ end of theoligonucleotide. The biotin group may be covalently linked to the5′-phosphate of the primer via a (CH₂)₆ spacer. Biotin-labeled primersmay be allowed to hybridize to the ssDNA template molecules preparedfrom the pBluescript® vector. The primer-template complexes may beattached to streptavidine coated beads according to Done et al.(Bioimaging 5: 139-152, 1997). At appropriate DNA dilutions, a singleprimer-template complex is attached to a single bead. A bead containinga single primer-template complex may be inserted into the reactionchamber 1501 of a sequencing apparatus 1500.

The primer-template may be incubated with modified T7 DNA polymerase(United States Biochemical Corp., Cleveland, Ohio). The reaction mixturemay contain unlabeled deoxyadenosine-5′triphosphate (dATP) anddeoxyguanosine-5′-triphosphate (dGTP), digoxigenin-labeleddeoxyuridine-5-triphosphate (digoxigenin-dUTP) and rhodamine-labeleddeoxycytidine-5′-triphosphate (rhodamine-dCTP). The polymerizationreaction may be allowed to proceed for 2 hours at 37° C. After synthesisof the digoxigenin and rhodamine labeled nucleic acid, the templatestrand may be separated from the labeled nucleic acid, and the templatestrand, DNA polymerase and unincorporated nucleotides washed out of thereaction chamber 1501. Alternatively, all deoxynucleoside triphosphatesused for polymerization may be unlabeled. In other alternatives, singlestranded nucleic acids may be directly sequenced without polymerizationof a complementary strand.

Exonuclease activity may be initiated by addition of exonuclease m tothe reaction chamber 1501. The reaction mixture may be maintained at pH8.0 and 37° C. As nucleotides 1510 are released from the 3′ end of thenucleic acid, they may be transported by microfluidic flow down themicrofluidic channel 1502. At the entrance to the microchannel 1503, anelectrical potential gradient created by a pair of electrodes 1504, 1505may be used to drive the nucleotides 1510 out of the microfluidicchannel 1502 and into the microchannel 1503. As the nucleotides 1510pass through the nanoparticles 1511, or alternatively into a resonantchamber, they may be exposed to excitatory radiation from a laser 1506.Raman emission spectra may be detected by the Raman detector 1507 asdisclosed below.

Raman Detection of Nucleotides

A Raman detection unit as disclosed in Example 2 may be used. The Ramandetector 1507 may be capable of detecting and identifying singlenucleotides 1510 of dATP, dGTP, rhodamine-dCTP and digoxigenin-dUTPmoving past the detector 1507. Data on the time course for labelednucleotide detection may be compiled and analyzed to obtain the sequenceof the nucleic acid. In alternative embodiments, the detector 1507 maybe capable of detecting and identifying single unlabeled nucleotides.

Example 2 Raman Detection of Nucleotides

Methods and Apparatus

In a non-limiting example, the excitation beam of a Raman detection unitwas generated by a titanium: sapphire laser (Mira by Coherent) at anear-infrared wavelength (750-50 nm) or a gallium aluminum arsenidediode laser (PI-ECL series by Process Instruments) at 785 nm or 830 nm.Pulsed laser beams or continuous beams were used. The excitation beamwas transmitted through a dichroic mirror (holographic notch filter byKaiser Optical or a dichromatic interference filter by Chroma or OmegaOptical) into a collinear geometry with the collected beam. Thetransmitted beam passed through a microscope objective (Nikon LUseries), and was focused onto the Raman active substrate, or into theresonant chamber, where target analytes (nucleotides or purine orpyrimidine bases) were located.

The Raman scattered light from the analytes was collected by the samemicroscope objective, and passed the dichroic mirror to the Ramandetector. The Raman detector comprised a focusing lens, a spectrograph,and an array detector. The focusing lens focused the Raman scatteredlight through the entrance slit of the spectrograph. The spectrograph(Acton Research) comprised a grating that dispersed the light by itswavelength. The dispersed light was imaged onto an array detector(back-illuminated deep-depletion CCD camera by RoperScientific). Thearray detector was connected to a controller circuit, which wasconnected to a computer for data transfer and control of the detectorfunction.

For surface-enhanced Raman spectroscopy (SERS), the Raman activesubstrate consisted of metal-coated nanostructures. Silvernanoparticles, ranging in size from 5 to 200 nm, were made by the methodof Lee and Meisel (J. Phys. Chem., 86:3391, 1982). Alternatively,samples were mixed with metallic nanoparticles and were placed on analuminum substrate under the microscope objective. The followingdiscussion assumes a stationary sample on the aluminum substrate. Thenumber of molecules detected was determined by the optical collectionvolume of the illuminated sample and the average concentration of theilluminated sample.

Single nucleotides may also be detected by SERS using microfluidicchannels. In various embodiments of the invention, nucleotides may bedelivered to a Raman active substrate through a microfluidic channel(between about 5 and 200 μm wide). Microfluidic channels can be made bymolding polydimethylsiloxane (PDMS), using the technique disclosed inAnderson et al. (“Fabrication of topologically complex three-dimensionalmicrofluidic systems in PDMS by rapid prototyping,” Anal. Chem.72:3158-3164, 2000).

Where SERS was performed in the presence of silver nanoparticles, thenucleotide, purine or pyrimidine analyte was mixed with LiCl (90 μMfinal concentration) and nanoparticles (0.25 M final concentrationsilver atoms). SERS data were collected using room temperature analytesolutions.

Results

Nucleoside monophosphates, purines and pyrimidines were analyzed bySERS, using the system disclosed above. Table 1 shows exemplarydetection limits for various analytes of interest.

TABLE 1 SERS Detection of Nucleoside Monophosphates, Purines andPyrimidines Number of Molecules Analyte Final Concentration DetecteddAMP 9 picomolar (pM) ~1 molecule Adenine  9 pM ~1 molecule dGMP 90 μM 6× 10⁶ Guanine 909 pM  60 dCMP 909 μM  6 × 10⁷ Cyotosine 90 nM 6 × 10³dTMP  9 μM 6 × 10⁵ Thymine 90 nM 6 × 10³

Conditions were optimized for adenine nucleotides only. LiCl (90 μMfinal concentration) was determined to provide optimal SERS detection ofadenine nucleotides. Detection of other nucleotides may be facilitatedby use of other alkali-metal halide salts, such as NaCl, KCl, RbCl orCsCl. The claimed methods are not limited by the electrolyte solutionused, and it is contemplated that other types of electrolyte solutions,such as MgCl₂, CaCl₂, NaF, KBr, LiI, etc. may be of use. The skilledartisan will realize that electrolyte solutions that do not exhibitstrong Raman signals will provide minimal interference with SERSdetection of nucleotides. The results demonstrate that the Ramandetection system and methods disclosed above were capable of detectingand identifying single molecules of nucleotides and purine bases. Thisdemonstrates Raman detection of unlabeled nucleotides at the singlenucleotide level.

Example 3 Raman Emission Spectra of Nucleotides, Purines and Pyrimidines

The Raman emission spectra of various analytes of interest was obtainedusing the protocol of Example 2, with the indicated modifications. FIG.16 shows the Raman emission spectra of a 100 mM solution of each of thefour nucleoside monophosphates, in the absence of surface enhancementand without Raman labels. No LiCl was added to the solution. A 10 seconddata collection time was used. Lower concentrations of nucleotides maybe detected with longer collection times, with surface enhancement,using labeled nucleotides and/or with added electrolyte solution.Excitation occurred at 514 nm. For each of the following figures, a 785nm excitation wavelength was used. As shown in FIG. 16, the unenhancedRaman spectra showed characteristic emission peaks for each of the fourunlabeled nucleoside monophosphates.

FIG. 17 shows the SERS spectrum of a 1 nm solution of guanine, in thepresence of LiCl and silver nanoparticles. Guanine was obtained fromdGMP by acid treatment, as discussed in Nucleic Acid Chemistry, Part 1,L. B. Townsend and R. S. Tipson (eds.), Wiley-Interscience, New York,1978. The SERS spectrum was obtained using a 100 msec data

FIG. 18 shows the SERS spectrum of a 10 nM cytosine solution, obtainedfrom dCMP by acid hydrolysis. Data were collected using a 1 secondcollection time.

FIG. 19 shows the SERS spectrum of a 100 nM thymine solution, obtainedby acid hydrolysis of dTMP. Data were collected using a 100 mseccollection time.

FIG. 20 shows the SERS spectrum of a 100 pM adenine solution, obtainedby acid hydrolysis of dAMP. Data were collected for 1 second.

FIG. 21 shows the SERS spectrum of a 500 nM solution of dATP (lowertrace) and fluorescein-labeled dATP (upper trace). dATP-fluorescein waspurchased from Roche Applied Science (Indianapolis, Ind.). The Figureshows a strong increase in SERS signal due to labeling with fluorescein.

Example 4 Resonance Enhanced Raman Detection of Single Molecules

This example demonstrates that the energies of Raman scattered lightgenerated from single molecules in resonant chambers may be detected byvarious known radiation detection devices. The results are based onsimulations performed by the inventors using MATLAB® available from TheMathWorks, Inc. of Natick, Mass.

FIG. 22 shows a plot comparing the energies of Raman scattered lightgenerated from single molecules respectively contained inside an opticalresonance chamber (curves A and B), or positioned in free space withoutresonance enhancement (curve C), according to one embodiment of theinvention. The energies, in Joules, are plotted on the y-axis, againsttime, in seconds, on the x-axis.

The curves A and B plot the energy of Raman scattered light stored inthe optical resonance chamber as a function of time. For both curves,the optical resonance chamber is capable of resonating the excitationlight as well as the Raman scattered light in order to provide evengreater resonant enhancement and stronger signals for detection. Thesimulations assumed a cavity quality factor of about 10⁶ for bothexcitation light and Raman scattered light. Even higher cavity qualityfactors are known in the arts. For example, cavity factors of about 10⁹have been reported by Spillane et al., in Nature, 415:621-623 (2002).The simulations assumed a Raman cross-section of about 10⁻²⁹ cm². Theexcitation light was provided as a 1 W continuous-wave. The time stepswere 100 nanoseconds.

Different hypothetical molecules with widely differing Raman gaincoefficients were used to generate the curves A and B. The Raman gaincoefficients essentially quantify the molecules likelihood of generatingstimulated Raman emissions. The curve A is based on a molecule with arelatively high Raman gain coefficient of about 20×10⁻⁸ cm/W, which istypical for silicon. The curve B is based on a molecule with zero Ramangain coefficient. As such, the curves A and B respectively provide nearupper and lower bounds for the stimulated Raman behavior of mostmolecules, including nucleic acid derivatives. The energy of thecumulative Raman scattered light generated during the given period oftime is plotted in curve C. There is no resonance enhancement in curveC.

As shown, the energies of Raman scattered light generated from thesingle molecules in the resonant chambers are significantly greater thanthe energy of the molecule positioned in free space (without resonanceenhancement). The greatest energies were observed when the stimulatedRaman process was induced (curve A). The energies for the stimulatedRaman (curve A) were around eight orders of magnitude larger than theenergies without resonance (curve C). Even when the stimulated Ramanprocess was not induced, the energies for the resonated spontaneousRaman process (curve B) were still about six orders of magnitude largerthan the energies without resonance (curve C). Such enhancement in theenergy available for detection is quite significant.

A dashed line at 10⁻²⁰ J indicates a typical detection level for avariety of commercially available high-quality radiation detectiondevices. Avalanche photodiodes, photomultiplier tubes, and intensifiedcharge-coupled devices typically have at least such sensitivities. TheRaman scattered light generated inside the optical resonance chamber inboth curves A and B exceed the indicated detection level within about100 microseconds. In contrast, the energy of Raman scattered light fromthe molecule in free space without optical resonance (curve C), remainsbelow the detection level for more than one second.

Accordingly, as demonstrated by these simulations, the energies of Ramanscattered light generated from single molecules in resonant chambers maybe detected by various known radiation detection devices. Significantly,this is true for both spontaneous Raman (curve B) and stimulated Raman(Curve A).

Example 5 Resonance Enhanced Raman Detection of Multiple Molecules

This prospective example demonstrates how to achieve even greaterdetection energies than those reported in Example 4 by using a pluralityof molecules, instead of just a single molecule. In this approach, aplurality of molecules may be introduced into the resonant chamber. Forexample, at least 100, or at least 1000 molecules are introduced intothe resonant chamber. In one aspect, enough molecules may be introducedto allow detection of a particular type of molecule with a desiredradiation detection device. To aid in identification, it may beappropriate to include a majority, or a vast majority, of the moleculesbeing of the same type.

The total detection energy generally increases for each additionalmolecule in the resonant chamber. Accordingly, each additional moleculeshould further increase the total energy of Raman scattered light in theresonant chamber. Often, the total energy of Raman scattered lightgenerated in the resonant chamber may initially increase substantiallylinearly for small numbers of molecules, and then increase even moredramatically (nonlinearly) for larger numbers of molecules. In this way,a plurality of molecules may be used to provide even stronger Ramanemission signals.

Thus, spectroscopic analysis chambers and methods for analyzing sampleswithin those chambers are disclosed. While the invention has beendescribed in terms of several embodiments, those skilled in the art willrecognize that the invention is not limited to the embodimentsdescribed, but may be practiced with modification and alteration withinthe spirit and scope of the claims. It is to be realized that theoptimum dimensional relationships for the parts of the invention, toinclude variations in size, materials, shape, form, function and mannerof operation, assembly and use, are deemed readily apparent to one ofordinary skill in the art, and all equivalent relationships to thoseillustrated in the drawings and described in the specification areintended to be encompassed by the present invention. The description isthus to be regarded as illustrative instead of limiting.

1-21. (canceled)
 22. An apparatus for nucleic acid sequencingcomprising: a reaction chamber having an immobilization surface forattachment of a nucleic acid molecule to be sequenced; a microfluidicchannel coupled to the reaction chamber for transporting nucleotidesreleased from the nucleic acid molecule; a microchannel coupled to themicrofluidic channel at an entrance for receiving the nucleotides, aregion of nanoparticles positioned within the microchannel, the regionof nanoparticles being configured to allow passage of the nucleotides;an excitation source proximate the region of nanoparticles, theexcitation source being configured to excite the nucleotides passingthrough the region of nanoparticles; and a detector configured to detectthe excited nucleotides.
 23. The apparatus of claim 22, furthercomprising a pair of electrodes configured to create an electricalpotential gradient proximate the entrance to drive the nucleotides fromthe microfluidic channel into the microchannel.
 24. The apparatus ofclaim 22, wherein the nanoparticles are cross-linked.
 25. The apparatusof claim 22, wherein the nanoparticles are gold nanoparticles.
 26. Theapparatus of claim 22, wherein the nanoparticles are silvernanoparticles.
 27. The apparatus of claim 22, wherein the size of thenanoparticles are from 5 nm to 60 mm.
 28. The apparatus of claim 22,wherein the excitation source is a laser.